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Porcine transforming growth
factor β1 （TGF-β1）ELISA Kit
（96 T）
? This immunoassay kit allows for the in vitro quantitative determination of porcine
TGF-β1 concentrations in serum, plasma and Cell Culture Supernates.
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to TGF-β1. Standards or samples are then added to
the appropriate microtiter plate wells with a biotin-conjugated
antibody preparation specific for TGF-β1 and Avidin conjugated to
Horseradish Peroxidase （HRP） is added to each microplate well and
incubated. Then a TMB （3,3',5,5' tetramethyl-benzidine） substrate
solution is added to each well. Only those wells that contain TGF-β1,
biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit
a change in color. The enzyme-substrate reaction is terminated by
the addition of a sulphuric acid solution and the color change is
measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.
The concentration of TGF-β1 in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
0.78 ng/ml-50 ng/ml. The standard curve concentrations used for the
ELISA’s were 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.12 ng/ml,
1.56 ng/ml, 0.78 ng/ml.
SPECIFICITY
This assay recognizes porcine TGF-β1. No significant cross-reactivity
or interference was observed.
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SENSITIVITY
The minimum detectable dose of porcine TGF-β1 is typically less
than 0.2 ng/ml.
The sensitivity of this assay, or Lower Limit of Detection （LLD） was
defined as the lowest protein concentration that could be
differentiated from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 2
Sample Diluent 1 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120μl
HRP-avidin 1 x 120μl
Wash Buffer
1 x 20 ml
（25×concentrate）
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
1 N HCI
1 x 10 ml
（May be stored for up to 1
month at room temperature）
1.2 N NaOH/0.5 M HEPES
1 x 10 ml
（May be stored for up to 1
month at room temperature）
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STORAGE
1. Unopened test kits should be stored at 2-8?C upon receipt and the
microtiter plate should be kept in a sealed bag. The test kit may be
used throughout the expiration date of the kit, provided it is stored
as prescribed above. Refer to the package label for the expiration
date.
2. Opened test plate should be stored at 2-8?C in the aluminum foil
bag with desiccants to minimize exposure to damp air. The kits will
remain stable until the expiring date shown, provided it is stored as
prescribed above.
3. A microtiter plate reader with a bandwidth of 10 nm or less and an
optical density range of 0-3 OD or greater at 450nm wavelength is
acceptable for use in absorbance measurement.
REAGENT PREPARATION
Bring all reagents to room temperature before use.
1. Wash Buffer If crystals have formed in the concentrate, warm
up to room temperature and mix gently until the crystals have
compley dissolved. Dilute 20 ml of Wash Buffer Concentrate
into deionized or distilled water to prepare 500 ml of Wash Buffer.
2. Biotin-antibody Centrifuge the vial before opening. Dilute to the
working concentration using Biotin-antibody Diluent（1:100）,
respectively.
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3. HRP-avidin Centrifuge the vial before opening. Dilute to the
working concentration using HRP-avidin Diluent（1:100）,
respectively.
4. Standard Centrifuge the standard vial at 6000-10000rpm for
30s. Reconstitute the Standard with 1.0 ml of Sample Diluent.
This reconstitution produces a stock solution of 50 ng/ml. Allow
the standard to sit for a minimum of 15 minutes with gentle
agitation prior to making serial dilutions. The undiluted standard
serves as the high standard （50 ng/ml）. The Sample Diluent
serves as the zero standard （0 ng/ml）. Prepare fresh for each
assay. Use within 4 hours and discard after use.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye,
hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450 nm,
with the correction wavelength set at 540 nm or 570 nm.
? Pipettes and pipette tips.
? Deionized or distilled water.
? Squirt bottle, manifold dispenser, or automated microplate
washer.
? An incubator which can provide stable incubation conditions up to
37°C±0.5°C.
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SAMPLE COLLECTION AND STORAGE
? Serum Use a serum separator tube （SST） and allow samples to
clot for 30 minutes before centrifugation for 15 minutes at 1000 g.
Remove serum and assay immediay or aliquot and store
samples at -20°C. Centrifuge the sample again after thawing
before the assay. Avoid repeated freeze-thaw cycles.
? Plasma Collect plasma using citrate, EDTA, or heparin as an
anticoagulant. Centrifuge for 15 minutes at 1000 g within 30
minutes of collection. Assay immediay or aliquot and store
samples at -20°C. Centrifuge the sample again after thawing
before the assay. Avoid repeated freeze-thaw cycles.
? Cell Culture Supernates Remove particulates by centrifugation
for 15 minutes at 1000 x g, 2 - 8°C and assay immediay or
aliquot and store samples at -20° C or -80℃. Avoid repeated
freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
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SAMPLE ACTIVATION PROCEDURE
To activate latent TGF-β1 to the immunoreactive form, prepare the
following solutions for acid activation and neutralization （provided in
our kit） .The solutions may be stored in polypropylene bottles at room
temperature for up to one month.
To activate latent TGF-β1 to immunoreactive TGF-β1 detectable by
the TGF-β1 ELISA assay, follow the activation procedure outlined
below. Assay samples after neutralization （pH 7.2 - 7.6）. Use
polypropylene test tubes.
Cell Culture Supernates Serum/Plasma
To 100μl of cell culture supernate,
add 20μl of 1 N HCI. Mix well.
To 80μl serum/plasma, add 20μl of 1
N HCl Mix well.
Incubate 10 minutes at room
temperature.
Incubate 10 minutes at room
temperature.
Neutralize the acidified sample by
adding 13 μl of 1.2 N NaOH/0.5 M
HEPES.
Neutralize the acidified sample by
adding 16μl of 1.2 N NaOH/0.5 M
HEPES.
Mix well. Mix well.
Assay immediay. Assay immediay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is recommended
that all samples, standards, and controls be assayed in duplicate. All the reagents
should be added directly to the liquid level in the well. The pipette should avoid
contacting the inner wall of the well.
1. Add 100μl of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 2 hours at 37°C.
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2. Remove the liquid of each well, don’t wash.
3. Add 100μl of Biotin-antibody working solution to each well.
Incubate for 1 hour at 37°C. Biotin-antibody working solution
may appear cloudy. Warm up to room temperature and mix gently
until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times
for a total of three washes. Wash: Fill each well with Wash Buffer
（200μl） and let it stand for 2 minutes, then remove the liquid by
flicking the plate over a sink. The remaining drops are removed by
patting the plate on a paper towel. Complete removal of liquid at
each step is essential to good performance.
5. Add 100μl of HRP-avidin working solution to each well. Cover the
microtiter plate with a new adhesive strip. Incubate for 1 hour at
37°C.
6. Repeat the aspiration and wash five times as step 4.
7. Add 90μl of TMB Substrate to each well. Incubate for 10-30
minutes at 37°C. Keeping the plate away from drafts and other
temperature fluctuations in the dark.
8. Add 50μl of Stop Solution to each well when the first four wells
containing the highest concentration of standards develop
obvious blue color. If color change does not appear uniform,
gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well within 30 minutes,
using a microplate reader set to 450 nm.
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CALCULATION OF RESULTS
Using the professional soft "Curve Exert 1.3" to make a standard curve is
recommended, which can be downloaded from our web.
Average the duplicate readings for each standard, control, and
sample and subtract the average zero standard optical density.
Create a standard curve by reducing the data using computer
software capable of generating a four parameter logistic （4-PL）
curve-fit. As an alternative, construct a standard curve by plotting the
mean absorbance for each standard on the x-axis against the
concentration on the y-axis and draw a best fit curve through the
points on the graph. The data may be linearized by plotting the log of
the TGF-β1 concentrations versus the log of the O.D. and the best fit
line can be determined by regression analysis. This procedure will
produce an adequate but less precise fit of the data. If samples have
been diluted, the concentration read from the standard curve must be
multiplied by the dilution factor.
LIMITATIONS OF THE PROCEDURE
? The kit should not be used beyond the expiration date on the kit
label.
? Do not mix or substitute reagents with those from other lots or
sources.
? It is important that the Standard Diluent selected for the standard
curve be consistent with the samples being assayed.
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? If samples generate values higher than the highest standard,
dilute the samples with the appropriate Standard Diluent and
repeat the assay.
? Any variation in Standard Diluent, operator, pipetting technique,
washing technique, incubation time or temperature, and kit age
can cause variation in binding.
? This assay is designed to eliminate interference by soluble
receptors, binding proteins, and other factors present in biological
samples. Until all factors have been tested in the Immunoassay,
the possibility of interference cannot be excluded.
TECHNICAL HINTS
? Centrifuge vials before opening to collect contents.
? When mixing or reconstituting protein solutions, always avoid
foaming.
? To avoid cross-contamination, change pipette tips between
additions of each standard level, between sample additions, and
between reagent additions. Also, use separate reservoirs for each
reagent.
? When using an automated plate washer, adding a 30 second soak
period following the addition of wash buffer, and/or rotating the
plate 180 degrees between wash steps may improve assay
precision.
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? To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary.
? Substrate Solution should remain colorless or light blue until
added to the plate. Keep Substrate Solution protected from light.
Substrate Solution should change from colorless or light blue to
gradations of blue.
? Stop Solution should be added to the plate in the same order as
the Substrate Solution. The color developed in the wells will turn
from blue to yellow upon addition of the Stop Solution. Wells that
are green in color indicate that the Stop Solution has not mixed
thoroughly with the Substrate Solution.